title: Proteomic analysis of the SH2 domain-containing leukocyte protein of 76 kDa (SLP76) interactome in resting and activated primary mast cells [corrected].
abstract: We report the first proteomic analysis of the SLP76 interactome in resting and activated primary mouse mast cells. This was made possible by a novel genetic approach used for the first time here. It consists in generating knock-in mice that express signaling molecules bearing a C-terminal tag that has a high affinity for a streptavidin analog. Tagged molecules can be used as molecular baits to affinity-purify the molecular complex in which they are engaged, which can then be studied by mass spectrometry. We examined first SLP76 because, although this cytosolic adapter is critical for both T cell and mast cell activation, its role is well known in T cells but not in mast cells. Tagged SLP76 was expressed in physiological amounts and fully functional in mast cells. We unexpectedly found that SLP76 is exquisitely sensitive to mast cell granular proteases, that Zn(2+)-dependent metalloproteases are especially abundant in mast cells and that they were responsible for SLP76 degradation. Adding a Zn(2+) chelator fully protected SLP76 in mast cell lysates, thereby enabling an efficient affinity-purification of this adapter with its partners. Label-free quantitative mass spectrometry analysis of affinity-purified SLP76 interactomes uncovered both partners already described in T cells and novel partners seen in mast cells only. Noticeably, molecules inducibly recruited in both cell types primarily concur to activation signals, whereas molecules recruited in activated mast cells only are mostly associated with inhibition signals. The transmembrane adapter LAT2, and the serine/threonine kinase with an exchange factor activity Bcr were the most recruited molecules. Biochemical and functional validations established the unexpected finding that Bcr is recruited by SLP76 and positively regulates antigen-induced mast cell activation. Knock-in mice expressing tagged molecules with a normal tissue distribution and expression therefore provide potent novel tools to investigate signalosomes and to uncover novel signaling molecules in mast cells.
authors.affiliation: Institut Pasteur, Département d'Immunologie, Unité d'Allergologie Moléculaire et Cellulaire, and Centre d'Immunologie Humaine Paris, France;
authors.forename: Yacine
authors.initials: Y
authors.lastname: Bounab
authors.forename: Anne-Marie-
authors.initials: AM
authors.lastname: Hesse
authors.forename: Bruno
authors.initials: B
authors.lastname: Iannascoli
authors.forename: Luca
authors.initials: L
authors.lastname: Grieco
authors.forename: Yohann
authors.initials: Y
authors.lastname: Couté
authors.forename: Anna
authors.initials: A
authors.lastname: Niarakis
authors.forename: Romain
authors.initials: R
authors.lastname: Roncagalli
authors.forename: Eunkyung
authors.initials: E
authors.lastname: Lie
authors.forename: Kong-Peng
authors.initials: KP
authors.lastname: Lam
authors.forename: Caroline
authors.initials: C
authors.lastname: Demangel
authors.forename: Denis
authors.initials: D
authors.lastname: Thieffry
authors.forename: Jérôme
authors.initials: J
authors.lastname: Garin
authors.forename: Bernard
authors.initials: B
authors.lastname: Malissen
authors.forename: Marc
authors.initials: M
authors.lastname: Daëron
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002478:Cells, Cultured; D008407:Mast Cells / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010750:Phosphoproteins / Q000378:metabolism*; D060066:Protein Interaction Maps; D040901:Proteomics; D051562:Proto-Oncogene Proteins c-bcr / Q000235:genetics / Q000378:metabolism; D017455:Receptors, IgE / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D010750:Phosphoproteins; D017455:Receptors, IgE; C092947:SLP-76 signal Transducing adaptor proteins; C496614:Bcr protein, mouse; D051562:Proto-Oncogene Proteins c-bcr